Anxiety disorders are the most prevalent psychiatric disorders and affect over 40 million American adults over the age of 18 every year. Significantly, ~ 20% of individuals who seek treatment for independent anxiety disorders also have a current drug use disorder. The extended amygdala is thought to play a critical role in adaptive motivational behavior, and has been implicated in the pathophysiology of maladaptive fear, anxiety, and addiction. Two key elements of the extended amygdala are the central nucleus of the amygdala (CeA) and the bed nucleus of the stria terminalis (BNST). Notably, evidence from clinical and preclinical studies suggests that differences in BNST activity determine individual differences in trait anxiety levels and also anticipatory anxiety (AA). Activity of corticotropin release factor (CRF) containing neurons in the BNST plays a central role in the normal adaptive response to stress. However, chronic release of CRF also plays a critical role in several psychopathologies, including anxiety disorders, posttraumatic stress disorder (PTSD), stress-induced drug recidivism, all of which have excessive AA as a core symptom. To date the cellular mechanisms underlying the switch from a normal adaptive response to a psychopathological state remain unknown. However, evidence from imaging studies suggests that activation of a circuit comprising the insular cortex (IC), amygdala, BNST, and ventrolateral periaqueductal grey (vlPAG) plays a key role in regulating the expression of AA. Using a transgenic CRF-Cre mouse line, we now have exciting pilot data showing that cell type-selective inhibition of CRF neurons in the BNST blocks the development of AA. The proposed work will use a multidisciplinary approach to define a functional circuit by testing the hypothesis that BNSTov CRF neurons act to integrate viscerosensory and emotional information from the IC and amygdala and relay this information to the vlPAG to regulate the expression of AA. The long-term objectives of this proposal are to delineate the cellular mechanisms contributing to the pathological switch in BNST function, with the hope of identifying novel targets for clinical intervention. Three Specific Aims will test the hypothesis: Aim 1 will examine the necessity / sufficiency of BNSTov CRF neuron activation in the expression of AA. Aim 2 will examine the role of the insular cortex (IC) and posterior basolateral amygdala (BLAp) in activating BNSTov CRF neurons during AA and their downstream connection with the vlPAG, and Aim 3 will examine the effects of chronic stress on the functioning of the proposed pathway.